1. Field of the Invention
Embodiments of the present invention relate to a nano-scale resolution microscopy system and methods of obtaining super-resolution images using the nano-scale resolution microscopy system, and more particularly, to methods and microscopy systems, capable of observing specimen at very high resolution by exploiting emission statistics of fluorescence probes excited by speckle illumination.
2. Description of the Related Art
A conventional optical microscope has a fundamental spatial resolution limit dependent on a wavelength of light and the numerical aperture of a lens. The best resolution of a conventional optical microscope corresponds to about a half of a wavelength, which refers to a diffraction limit.
Fluorophores, such as fluorescence probes or fluorescence proteins have been extensively designed to be specific to particular cellular functions such as signal transduction and gene expression, so fluorescence microscopy has become an invaluable tool in biology.
In fluorescence microscopy, fluorophores are directly attached to a region of interest within a cell or particular proteins. However, the conventional microscopes may have a limitation in overcoming the diffraction limit of the microscope optical systems, and more particularly, a limitation in resolving fluorophores which are separately less than the diffraction limit.
To address this problem, super-resolution far-field fluorescence nanoscopy have been extensively investigated. This super-resolution microscopy is to exploit non-linear optical phenomena to break the diffraction limit. In STED microscopy, a Gaussian shape excitation beam and a red-shifted doughnut-shaped STED beam are used to quench excited fluorophores by stimulated emission from excitation to ground state. (Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proceedings of the National Academy of Sciences of the United States of America 97, 8206-8210 (2000).) In saturated structured illumination microscopy (SSIM), structured illumination is used to extend the spatial bandwidth of the optical system using the synthetic aperture principle. (Gustafs son, M. G. L. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proceedings of the National Academy of Sciences of the United States of America 102, 13081 (2005).) In PALM and STORM for each imaging cycle, an optically resolvable random subset of photoswitchable fluorophores is excited to the active state and then switched off to the dark state quickly using background quenching lights. (Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793-796 (2006).)